In the recent past, light-microscopy methods have been developed with which, based on a sequential, stochastic localization of individual point-like objects (in particular, fluorescent molecules), it is possible to display image structures that are smaller than the diffraction-related resolution limit of classic light microscopes. Such methods are described, for example, in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching,” Geisler C. et al., Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as “localization microscopy.”
The new methods have in common the fact that the structures to be imaged are prepared with markers that possess two distinguishable states, namely a “bright” state and a “dark” state. If fluorescent dyes are used as markers, for example, the bright state is then a fluorescence-capable state and the dark state is a non-fluorescence-capable state. In order for sample structures to be imaged at a resolution that is smaller than the classic resolution limit of the image-producing optical system, a small subset of the markers is then repeatedly prepared into the bright state. This subset is referred to hereinafter as an “active subset.” The active subset must be selected so that the average spacing between adjacent markers in the bright state—and thus the state capable of imaging by light microscopy—is greater than the resolution limit of the imaging optical system. The luminance signals of the active subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. A spot of light, whose size is determined by the resolution limit of the image-producing optical system, is thus acquired from each marker.
The result is that a plurality of individual raw-data images are acquired, in each of which a different active subset is imaged. In an image analysis process, the center points of the light distributions (representing the markers that are in the bright state) are then determined in each individual raw-data image. The center points of the light spots identified from the individual raw-data images are then combined into one overall depiction. The high-resolution overall image produced by this overall depiction reflects the distribution of the markers. For representative reproduction of the structure to be imaged, a sufficient number of signals must be detected. But because the number of markers in the particular active subset is limited by the minimum average spacing that must exist between two markers in the bright state, a very large number of individual raw-data images must be acquired in order to image the structure completely. The number of individual raw-data images is typically in a range from 10,000 to 100,000.
The time required for acquiring an individual raw-data image is limited at the low end by the maximum image acquisition rate of the imaging detector. This leads to comparatively long total acquisition times for a sequence of individual raw-data images that is necessary for the overall depiction. The total acquisition time can thus amount to as much as several hours.
In order to reduce the total acquisition time as much as possible and to keep it as short as possible, the acquisition time of each individual raw-data image must be minimized. This requires that the individual light spots that generate the markers be produced very quickly, although of course they must nevertheless be bright for the purpose of good localization. This means that when they are in the active state, the markers must emit a very large number of photons very quickly before they transition back into the inactive state. If the acquisition time of the individual raw-data image is then adapted in accordance with the average luminous time of the markers, the result is the fastest possible image acquisition. Typical markers are (switchable or non-switchable) fluorescing molecules. The more photons of excitation light they acquire, the more photons they emit within a certain time period. It is therefore necessary to irradiate the molecules with high light intensities, this being the only way to enable maximally fast image acquisition.
High light intensities are advantageous in particular when quite ordinary, non-switchable fluorescent molecules are used as markers. These have the advantage, as compared with special switchable dyes, of generally being much easier to handle and yielding better color results. In addition, a large number of different dyes are available. The number of special switchable dyes is limited, and often they are not easy to handle. The active state of these standard dyes thus corresponds to the molecule in its ground state or first excited state, in any event with the capability of emitting photons. The inactive state is a dark state of the molecule as exhibited by every ordinary fluorescent molecule, for example a triplet state or a reduced or oxidized state. The molecule can typically change with a low probability from the excited state into the dark state, and after a certain time will automatically transition back into the ground state in which it can fluoresce again. Because of this fact, the molecule exhibits blinking behavior: it emits photons until it spontaneously transitions out of the excited state into the dark state. The entirety of these photons forms a light spot. The molecule then remains in the dark state until it transitions spontaneously back into the active state. During the dark-state time the molecule emits no photons and thus no detectable signal. Because the dark state can be very long-lived, but because at high light intensity the light spots occur very quickly, this behavior manifests itself as blinking. In order to achieve, in these circumstances, a state in which only individual molecules are visible (i.e. are in the active state), the light intensity of the excitation light must be very high. Only in this way is it possible for almost all the molecules to be in the inactive state, since as soon as they spontaneously change into the active state they emit (because of the high intensity of the excitation light) a rapid sequence of photons and then disappear again “in a flash.” Only a very few molecules are therefore visible simultaneously. The high intensity of the excitation light here therefore not only makes possible rapid image acquisition, but is also useful for establishing a sufficiently low ratio of active to inactive markers.
It has become apparent from the explanations above that a high level of light output must be introduced into the sample. The sample is usually illuminated using the so-called “widefield” method. This means that a large area in the sample is illuminated as homogeneously as possible, specifically exactly that region which is imaged by the imaging optics onto the detector. Illumination typically occurs through the same objective that also collects the fluorescent light and images the sample onto the detector by means of a corresponding further optic.
In order to allow a large region of the sample to be illuminated as homogeneously as possible, light is focused into the rear focal plane of the objective. Depending on the size of the focus in the rear focal plane, what results in the front focal plane, i.e. in the sample plane, is the size of the illuminated field. The smaller the focus in the rear focal plane, the larger the illuminated field, and vice versa. In order to allow a correspondingly large region to be illuminated, all of the excitation light is therefore directed as a small light beam into the objective. The consequence of this is that in typical objectives, the excitation light passes through the lens elements in the objective as a relatively narrow ray bundle. There are regions in the objective in which the ray bundle is wider or narrower. In general, however, with this type of illumination the entire aperture of the lens elements is typically not even utilized, but instead the light always passes through the lens elements only in a very thin ray bundle. Since it is generally necessary, as already mentioned above, to introduce very high light power levels into the sample, extremely high light intensities are achieved as a result of the thin ray bundle in the objective. Laser light is therefore often used for widefield illumination in localization microscopes in order to achieve these light intensities.
More-complicated lens elements or lens systems, such as those that occur in objectives, typically contain various materials that can experience damage at high light intensities. The “weakest link” here is typically the optical cement with which, for example, different lens parts are joined to one another. In extreme cases, however, even surface layers on the lens elements, or the elements themselves, can be damaged.
As the “load” on the optic due to the strong illumination increases, the optic can thus suffer damage that can have a disadvantageous effect on the beam path of the illumination and on detection. “Blind spots” can occur, for example, producing point-like absorption, diffraction, or severe scattering of light. Holes in the elements, etc. are also conceivable. Such damage can make an objective completely unusable. For example, if a blind spot develops exactly in the illumination beam path (where it would preferentially develop as a result of the beam load), the objective would no longer be usable, since light (or at least a “clean” illumination beam) would no longer be able to emerge from the objective toward the sample. Damage of this kind would also have a negative effect on the detection beam path, since it too passes through the objective, and the detected light would be influenced (e.g. scattered) at the damaged sites in the objective.
Although it is known to manufacture simple optical systems without cement, these systems typically are mechanically more susceptible and less stable, and disadvantages are also conceivable in terms of other optical properties. These systems are appreciably more stable in terms of beam load, however, since the optical cement that is susceptible to beam load is omitted. With complicated optical systems such as special objectives, however, production without optical cement is not possible by present-day industrial standards, or is possible only with considerable extra outlay. It is thus extremely important to prevent such damage even in the context of cemented optical systems, since otherwise the service life of the objectives for practical use is greatly limited, the more so since objectives are very complex—and correspondingly expensive—components.
What emerges upon closer examination of the problem is that typical radiation damage proceeds according to a similar pattern. In most cases the damage does not occur immediately when the light is first switched on. The light power levels used are often ones that the problematic optical components can withstand for an extended time. A slow decrease in the transmittance of the optical system can nevertheless often be observed. In the case of cemented optics this is presumably attributable to a change in the cement under the influence of light. After a certain time a critical threshold is reached, however, and the questionable component exhibits a sudden change (in fractions of a second) at the illuminated site; the result of this can be that at the illuminated site and in its vicinity, the component can no longer transmit light at that point or (if the reaction is correspondingly severe) in its entirety, or it may exhibit severe light scattering and has thus become unusable.
Before this sudden change occurs, what is detectable is at most a marginal change in optical properties, which may be measurable with suitable and exact measurement methods but is usually negligible. The optic is thus usable in entirely normal fashion until the change suddenly occurs. The damage to or destruction of the objective is therefore a highly nonlinear process, and this is exploited in the invention below.
It is understood that the problem occurs not only with localization microscopes but also with any type of microscope that uses a high illumination light intensity which is sent through the objective. Although this is particularly relevant to fluorescence microscopes, it can also be the case with light microscopes.